CN107056909A - Application of the OsNPF5.11 genes in rice yield is improved - Google Patents

Abstract

The invention disclosesOsNPF5.11Application of the gene in rice yield is improved, belongs to plant genetic engineering field.OsNPF5.11The amino acid sequence of DNA encoding the protein is as shown in SEQ ID NO.1, and cDNA sequence is as shown in SEQ ID NO.2.The present invention is by building paddy riceOsNPF5.11Gene overexpression plant,OsNPF5.11Gene disturbs plant, finds by improvingOsNPF5.11Gene expression, can make normal rice tillering number, every plant of grouting grain number increase, thereforeOsNPF5.11Gene can be used in paddy rice seed selection improving rice yield.OsNPF5.11Gene has important application value in terms of Nitrogen effect plant growth and growth course is illustrated and in terms of plant type of rice improvement.

Description

OsNPF5.11Application of the gene in rice yield is improved

Technical field

The invention belongs to plant genetic engineering field, and in particular toOsNPF5.11Gene answering in rice yield is improved With.

Background technology

Plant obtains nitrogen by absorbing the ammonia in soil, nitrate anion, amino acid, soluble peptide etc.；The absorption of nitrogen and Transhipment relies primarily on ammonium root transport protein（AMT）, nitrate anion transport protein（NRT）, amino acid transport proteins（AAT）, peptide transport Albumen（PTR）Completed Deng transport protein（Williams L, Miller A. Transporters responsible for the uptake and partitioning of nitrogenous solutes. Annual Review of Plant Physiology and Plant Molecular Biology, 2001, 52: 659-688.）.Ammonium is absorbed by plant AMT Pass through glutamine synthelase again afterwards（GS）And glutamate synthase（GOGAT）Glutamine and glutamic acid are synthesized, the latter enters one again Step forms other amino acid（Sonoda Y, Ikeda A, Saiki S, et al. Feedback regulation of the ammonium transporter gene family AMT1 by glutamine in rice. Plant Cell Physiology, 2003, 44: 1396-1402.）.Plant can pass through high affine movement system（HATS）NRT2 and low parent And movement system（LATS）NRT1 absorb environment in nitrate, by nitrate reductase（NR）And nitrite reductase（NiR） Reduction forms ammonium, further forms amino acid（Paungfoo-Lonhienne C, Lonhienne T G, Rentsch D, et al. Plants can use protein as a nitrogen source without assistance from other organisms. Proceedings of the National Academy of Sciences, 2008, 105: 4524-4529.）.

Nitrogen transport NPF families include NRT1 and PTR subfamilies, and different members are in plant different parts transporting nitric acid root, widow Peptide or amino acid etc., serve different on vine growth and development（Rentsch D, Schmidt S, Tegeder M. Transporters for uptake and allocation of organic nitrogen compounds in plants. Febs Letters, 2007, 581: 2281-2289.）.OsNPF2.2 has mediated unloading for xylem nitrate anion Carry, influence paddy growth（Li Y, Ouyang J, Wang Y Y, et al. Disruption of the rice nitrate transporter OsNPF2.2 hinders root-to-shoot nitrate transport and vascular development. Scientific reports, 2015, 5: 9635.）.OsNPF7.2Have to nitrate anion Low affine transport, can influence plant strain growth（Hu R, Qiu D, Chen Y, et al. Knock-down of a tonoplast localized low-affinity nitrate transporter OsNPF7.2 affects rice growth under high nitrate supply. Frontiers in plant science, 2016, 7.）.

Although known nitrogen nutrition can promote growth and development of plants, nitrogen nutrition have impact on the what kind of growth of plant There is presently no the understanding of system for development.There is more than 80 member in other paddy rice NPF families, and nitrogen nutrition is by NPF gene families Any member is responded, and what position to have mediated nitrogen nutrition transport at, so as to have impact on, plant is what kind of to grow, It is current also almost unknown.So, nitrogen efficient transportation gene may be had by excavating in NPF families, particularly can control paddy rice The nitrogen transport key gene of plant type, is beneficial to the cultivation of rice high yield kind.The present invention after studying for a long period of time by having found, NPF FamilyOsNPF5.11Gene has two kinds of alternative splicing forms, and the first splicing form plays main function, had to rice tillering Important positive regulating and controlling effect.After sequence is by overexpression, the improvement of plant plant type is may be directly applied to, so that increasing production of rice.

The content of the invention

It is an object of the invention to solve problems of the prior art, there is provided paddy rice NPF gene family membersOsNPF5.11Application of the gene in rice yield is improved.

The purpose of the present invention is achieved through the following technical solutions：

The present invention is with the NPF gene family members of paddy riceOsNPF5.11Gene is object, is cloned in spending 11 from paddy riceOsNPF5.11CDNA sequence.By buildingOsNPF5.11Gene overexpression vector, using AgrobacteriumEHA105The something lost of mediation Method for transformation is passed, overexpression vector is imported and spent in normal japonica rice variety in 11, is obtainedOsNPF5.11Gene overexpresses plant, Its tiller number, grouting grain number are significantly improved compared with spending 11 in control wild type.Built by RNAi technologyOsNPF5.11Gene Interference expression vector, spends in 11 during interference expression vector is imported, obtainsOsNPF5.11The interference that gene expression amount declines is planted Strain, disturbs the tiller number of plant, grouting grain number to be significantly reduced compared with spending 11 in.These results indicate that by improvingOsNPF5.11The expression of gene, can make normal rice tillering number, grouting grain number increase, so as to improve rice yield.

Based on present invention discover thatOsNPF5.11The function of gene,OsNPF5.11Gene can be used in paddy rice seed selection.Institute The paddy rice seed selection stated is improves rice tillering number, grouting grain number, so as to improve rice yield.Specifically can be by improvingOsNPF5.11The expression of gene makes rice tillering number and every plant of grain number increase of being in the milk, and reaches the purpose for improving rice yield.

OsNPF5.11Gene can also be used for improving the yield of other plant, such as be made by transgenosisOsNPF5.11Gene exists In plant（Excess）Expression, to improve the branch quantity of plant, and then is improved the yield of plant.Described plant refers to Monocotyledon or dicotyledon；Such as：Wheat, tomato, turfgrass or clover etc..

DescribedOsNPF5.11The amino acid sequence of the OsNPF5.11 albumen of gene code is as shown in SEQ ID NO.1； DescribedOsNPF5.11The cDNA sequence of gene is preferably as shown in SEQ ID NO.2.

It is construed as, on the premise of OsNPF5.11 protein actives are not influenceed（I.e. not in the activated centre of albumen）, this Art personnel can carry out various substitutions, additions and/or deletions one or several to the amino acid sequence shown in SEQ ID NO.1 Individual amino acid obtains the amino acid sequence with equal function.Therefore, OsNPF5.11 albumen is also included shown in SEQ ID NO.1 Amino acid sequence is substituted, replaces and/or increased the protein having with isoreactivity that one or several amino acid are obtained.This Outside, it should be understood that, it is contemplated that the degeneracy of codon and the preferences of different plant species codon, those skilled in the art can root According to the codon for needing to use suitable particular species expression.

Advantages of the present invention and effect：

（1）What the present invention was clonedOsNPF5.11Make rice tillering number and grouting grain number increase, explanation after gene overexpressionOsNPF5.11Gene pairs improves rice yield and had a significant effect, and therefore, is improved by technique for gene engineeringOsNPF5.11Gene Expression can improve plant products.It is not only does this facilitate and cultivates high-yield rice under the conditions of nitrogen by normally applying, can also passes through Molecular breeding carries out the breed improvement of plant.

（2）OsNPF5.11The successful clone of gene, further demonstrate important work of the NPF families in nitrogen absorption process With, there is important meaning to the biological function for illustrating NPF families, in addition to further appreciating that plant nitrogen metabolism approach, raising Nitrogen absorption efficiency has great impetus.

（3）Although being cloned into some genes for improving plant products at present, to the molecular mechanism of plant yield-increasing still It is unclear.And what the present invention was clonedOsNPF5.11Gene can improve the yield of paddy rice, the key factor of pair determination plant yield-increasing There is great impetus.

Brief description of the drawings

Fig. 1 be spend 11 in control,OsNPF5.11Gene overexpress 2 strains of plant andOsNPF5.11Gene disturbs plant The whole strain phenotypic map of 2 strains.

Fig. 2 be spend 11 in control,OsNPF5.11Gene overexpress 2 strains of plant andOsNPF5.11Gene disturbs plant The statistics block diagram of 2 strain tiller numbers, data carry out variable analysis using SPSS softwares（ANOVA）, use Duncan ' s to exist Significance difference analysis, different group lowercases are carried out in 0.05 level（a、b、c）Represent significant difference.

Fig. 3 be spend 11 in control,OsNPF5.11Gene overexpress 2 strains of plant andOsNPF5.11Gene disturbs plant Every plant of seed grouting grain number figure of 2 strains.

Fig. 4 be spend 11 in control,OsNPF5.11Gene overexpress 2 strains of plant andOsNPF5.11Gene disturbs plant The statistics block diagram of every plant of seed grouting grain number of 2 strains, data carry out variable analysis using SPSS softwares（ANOVA）, use Duncan ' s carry out significance difference analysis, different group lowercases in 0.05 level（a、b、c）Represent significant difference.

Fig. 5 be spend 11 in control,OsNPF5.11Gene overexpress 2 strains of plant andOsNPF5.11Gene disturbs plant In 2 strainsOsNPF5.11The statistics block diagram of gene relative expression quantity, data carry out variable analysis using SPSS softwares （ANOVA）, use Duncan ' s to carry out significance difference analysis, different group lowercases in 0.05 level（a、b、c）Table Show significant difference.

Embodiment

With reference to embodiment, the present invention will be further described in detail, but the implementation of the present invention is not limited to this. Unless otherwise specified, the conventional meanses that the technological means used in following embodiments is well known to those skilled in the art；Used Experimental method is conventional method, and can be according to the recombinant technique described（Referring to molecular cloning, laboratory manual, second edition, CSH Press, Cold SpringHarbor, New York）Complete；Material, reagent used etc., are commercially obtained.

Embodiment 1OsNPF5.11Gene overexpresses the structure of plant

Extract in paddy rice and spend 11 RNA, and its reverse transcription is utilized into primer pair into cDNA：

F1：5'-GGTACCATGTCGGGGAAGAAGCCGACGGCG-3'（kpnI）,

R1：5'-TCTAGAGTAGTACTTGCACAAGTGTGTGAA-3'（XbaI）；

Expanded by PCROsNPF5.11After the cDNA of gene, pass throughkpnI、XbaPCAMBIA-1306 carriers are connected into after I digestions （PCAMBIA-1306 carriers are purchased from Cambia companies）, constructOsNPF5.11The overexpression vector of geneOsNPF5.11- p1306.Using AgrobacteriumEHA105The genetic transforming method of mediation, overexpression vector is imported in normal rice varieties and spends 11 In.

Obtained all transgenic plants are transplanted in the basket with soil, periodically watered, applies fertilizer, treats that seedling grows tall about During 10cm, plant in big Tanaka, after seedling is grown up, extract genomic DNA and detect that detection is drawn to transfer-gen plant by PCR Thing to for：

F2：5'-GATGTTGGCGACCTCGTATT-3',

R2：5'-TCGTTATGTTTATCGGCACTTT-3'.

If amplifying 517bp fragment, it is positive plant to illustrate transfer-gen plant.Positive plant individual plant sowing is simultaneously planted Plant, until T2 generations identify the transfer-gen plant of homozygosis, that is, obtainOsNPF5.11Gene overexpresses plant.OsNPF5.11Gene The tiller number of plant is overexpressed far more than spending 11 plant, significant difference, as shown in Fig. 1,2 in control.Individual plant collects seed, hair Now every plant of grouting grain number of overexpression plant, which is significantly more than in control, spends 11 plant, as shown in Fig. 3,4.

TakeOsNPF5.11Gene overexpresses plant leaf, extracts RNA and by its reverse transcription into cDNA, passes through real-time fluorescence Quantitative PCR detectionOsNPF5.11The expression quantity of gene, as a result shows（Fig. 5）Overexpress plantOsNPF5.11The expression quantity of gene The significantly rise compared with spending 11 in control.Real-time fluorescence quantitative PCR the primer to for：

F3：5'-AGGCTGAGCAGGGCATGCGTGA-3',

R3：5'-AGCATGGACGTCACCCCGCT-3'.

Embodiment 2OsNPF5.11Gene disturbs the structure of plant

Extract in paddy rice and spend 11 RNA, and its reverse transcription is utilized into primer pair into cDNA：

F4：5'-GGTACCAGGCTGAGCAGGGCATGCGTGA-3'（KpnI）,

R4：5'-GGATCCTGTAGCCGCCTTGGCCGATGGA-3'（BamHI）；

F5：5'-ACTAGTAGGCTGAGCAGGGCATGCGTGA-3'（SpeI）,

R5：5'-GAGCTCTGTAGCCGCCTTGGCCGATGGA-3'（Sac I）；

Respective PCR is amplifiedOsNPF5.11After the cDNA fragments of gene, by being connected into after corresponding digestion with restriction enzyme PTCK303 carriers, are constructedOsNPF5.11The interference expression vector of geneOsNPF5.11-pTCK303.Using AgrobacteriumEHA105The genetic transforming method of mediation, interference expression vector is imported and spent in normal japonica rice variety in 11.

Obtained all transgenic plants are transplanted in the basket with soil, periodically watered, applies fertilizer, treats that seedling grows tall about During 10cm, plant in big Tanaka, after seedling is grown up, extract genomic DNA and detect that detection is drawn to transfer-gen plant by PCR Thing to for：

F2：5'-GATGTTGGCGACCTCGTATT-3',

R2：5'-TCGTTATGTTTATCGGCACTTT-3'.

If amplifying 517bp fragment, it is positive plant to illustrate transfer-gen plant.Positive plant individual plant sowing is simultaneously planted Plant, until T2 generations identify the transfer-gen plant of homozygosis, that is, obtainOsNPF5.11Gene disturbs plant.OsNPF5.11Gene is done The tiller number of plant is disturbed far fewer than spending 11 plant, significant difference, as shown in Fig. 1,2 in control.Individual plant collects seed, finds dry Disturb every plant of plant grouting grain number and be considerably less than in control and spend 11 plant, as shown in Fig. 3,4.

TakeOsNPF5.11Gene disturbs plant leaf, extracts RNA and by its reverse transcription into cDNA, fixed by real-time fluorescence Measure PCR detections OsNPF5.11The expression quantity of gene, as a result shows（Fig. 5）Disturb plantOsNPF5.11The expression quantity of gene with Spending 11 to compare in control significantly reduces.Real-time fluorescence quantitative PCR the primer be the same as Example 1.

The above results show, by improvingOsNPF5.11The expression of gene, can increase the tiller number and grouting grain of paddy rice Number, and then improve rice yield.

Above-described embodiment is preferably embodiment, but embodiments of the present invention are not by above-described embodiment of the invention Limitation, other any Spirit Essences without departing from the present invention and the change made under principle, modification, replacement, combine, simplification, Equivalent substitute mode is should be, is included within protection scope of the present invention.

SEQUENCE LISTING

<110>Wuhan Bioengineering Institute

<120>Application of the OsNPF5.11 genes in rice yield is improved

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<160> 2

<170> PatentIn version 3.3

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<211> 535

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<213> Oryza sativa

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Gly Leu Ile Gly Leu Thr Leu Trp Ala Leu Leu His Thr Arg Met Pro

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Gly Tyr Asn Pro Ser Leu Gln Ala Phe Gly Ala Asp Gln Leu Asp Ile

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Gly Asp Asp Asp Asp Asp Gly Asp Asn Gly Ala Thr Ala Ala Thr Glu

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Glu Gln Arg Ser Lys Val Lys Ser Leu Phe Phe Gln Trp Trp Tyr Phe

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Gly Ile Cys Ser Gly Ser Leu Leu Gly Asn Thr Thr Met Ser Tyr Val

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Gln Asp Thr Val Gly Trp Gly Leu Gly Phe Ala Val Pro Ala Ala Val

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Met Ala Val Ser Val Ala Ala Phe Phe Cys Cys Thr Pro Leu Tyr Lys

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Gln Arg Gln Pro Arg Ala Val His Arg Lys Pro Cys Arg Asp Ser Val

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Leu Lys Ala Leu Lys Ser Leu Leu Ala Ser Val Thr Gly Ala Arg Lys

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Ile Thr Leu Pro Ser Arg Asp Gly Asp Asp Asp Thr Asp Ile Val Ser

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Glu Leu Glu Leu Gln Glu Lys Pro Leu Lys Leu Ala Asp Gln Lys Gln

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Glu Ala Ala Met Gly Glu Ala Ala Ala Pro Ser Val Ala Lys Ile Ile

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Val Arg Leu Leu Pro Ile Trp Thr Met Leu Leu Met Phe Ala Val Ile

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atgatcatag tggtggcgag cgtggagagg ttcgcgtaca agggggtggc gtcgaacctg 120

gtgacgtacc tgacggaggt ggtggagatg agcacgtcgg cggcggcgaa gagcgtcagc 180

gcgtggagcg gggtgacgtc catgctgccg ctcctcaccg ccgtcctcgc cgactcctac 240

tgggaccgct actccaccat caccgcctcc tccctcctct acgtcgtcgg gctaataggc 300

ctaactttat gggcactgct acacacacgg atgccatgct ccacgctctt cttcccgctc 360

tacctcatct ccatcggcca aggcggctac aacccttcgc tgcaagcctt cggcgccgac 420

cagctcgaca tcggcgacga cgacgatgac ggcgacaatg gcgcaacagc agcaacagaa 480

gagcagagga gcaaggtgaa gagcctcttc ttccagtggt ggtacttcgg catctgcagc 540

ggcagcctgc tggggaacac gacaatgtcg tacgtccagg acaccgtcgg ctggggcctc 600

ggcttcgccg tcccggccgc cgtcatggcc gtctccgtcg ccgccttctt ctgctgcacc 660

cccctctaca agcagaggca acccagagct gttcatcgca agccctgtcg tgacagcgtc 720

ctcaaagccc tgaaatcgct tctcgcaagt gtcactggcg ccagaaagat caccctgcca 780

tccagagacg gcgacgatga cactgacatc gtatctgagc tagagttgca ggagaagcca 840

ctgaagctgg ctgatcagaa gcaggaggcg gccatgggag aggctgcagc accaagtgta 900

gccaagatca tagtgaggct gctcccaatc tggacgatgc tgctcatgtt cgcggtcatc 960

ttccagcagc cgatgacgtt cttcaccaag caggggatgc tgatggacca ccgcgtcggc 1020

gccgtgttcg tgatcccccc ggcgatgctg cagagctcca tcaccgtctc catcatcctc 1080

ctcatgccgc tctacgacac ggtggtggtg ccgctcgccg gcctcgtcgc cggccacggc 1140

aaggggatca cggtgctcca gcggatcggc gtcgggatgg tgctctccat cgtcgccatg 1200

gcggtcgccg cgctcgtcga ggcgcgccgc ctccgcgccg ccgcgtcgtc ctcgtccggc 1260

ggccgcctga gcatattctg gctcctcccg cagtacgtgc tcctcggcgt ctccgacgtg 1320

ttcacggtgg tgggcatgca ggagttcttc tacacccagg tccccagcgc catgaggacc 1380

gtcggcatcg cgctctacct cagcgtcttc ggcgtcggca gcttcgtcgg cgcgttcctc 1440

atcaccgcgc tcgagatggt gacggcgggg ggcggcggcg gcgggcacga tcacgggtgg 1500

ttctccgatg atccccggga ggcgcggctg gacaagtact actggttctt ggcgctcctc 1560

agctgcgtca gcttcgtcgt cttcacacac ttgtgcaagt actactag 1608

Claims (9)

1.OsNPF5.11Application of the gene in rice tillering number is improved, it is characterised in that：By improvingOsNPF5.11Gene Expression make rice tillering number increase.

2.OsNPF5.11Application of the gene in rice milking stage grain number is improved, it is characterised in that：By improvingOsNPF5.11Base The expression of cause makes the increase of rice milking stage grain number.

3. OsNPF5.11Application of the gene in rice yield is improved, it is characterised in that：By improvingOsNPF5.11Gene Expression improves rice yield.

4. OsNPF5.11Application of the gene in paddy rice seed selection.

5.OsNPF5.11Application of the gene in plant branching number and yield is improved.

6. application according to claim 5, it is characterised in that：Described plant refers to monocotyledon or dicotyledon.

7. the application according to claim 5 or 6, it is characterised in that：Described plant include wheat, tomato, turfgrass or Clover.

8. the application according to claim any one of 1-5, it is characterised in that：DescribedOsNPF5.11Gene code The amino acid sequence of OsNPF5.11 albumen is as shown in SEQ ID NO.1；Or OsNPF5.11 albumen is ammonia shown in SEQ ID NO.1 Base acid sequence is substituted, replaces and/or increased the protein having with isoreactivity that one or several amino acid are obtained.

9. application according to claim 8, it is characterised in that：DescribedOsNPF5.11The cDNA sequence of gene such as SEQ Shown in ID NO.2.